In accordance with the present invention, novel monomeric and multimeric compounds having enhanced relaxivities are provided. These compounds are useful, for example, as metal-chelating ligands. The compounds are also useful in the form of metal complexes as diagnostic contrast agents. When the metal in the complex is paramagnetic, the diagnostic contrast agents are especially suitable for magnetic resonance imaging (MRI).
In one embodiment of the invention, certain specific compounds comprise a tetraazacyclododecane macrocycle, and are represented by the formula I: 
wherein
each m, n, o and p is independently 1 or 2;
q is 0 or 1;
each G is independently xe2x80x94COORxe2x80x3, xe2x80x94P(O)(ORxe2x80x3)2, xe2x80x94P(O)(ORxe2x80x3)(Rxe2x80x3) or xe2x80x94C(O)N(Rxe2x80x3)2;
each Rxe2x80x2 is independently hydrogen or alkyl, alkoxy, cycloalkyl, hydroxyalkyl or aryl, each of which is optionally substituted, or a functional group capable of forming a conjugate with a biomolecule or of forming a multimer of said compound of formula I;
each Rxe2x80x3 is hydrogen;
each R13 through R20 is independently hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl or a functional group capable of forming a conjugate with a biomolecule or of forming a multimer of said compound of the formula I;
or R13 together with R15, and R17 together with R18, independently form, together with the carbon atoms in the tetraazacyclododecane macrocycle to which they are attached, a fused fully or partially saturated non-aromatic cyclohexyl ring which may be unsubstituted or substituted by one or more halogen, alkyl, ether, hydroxy or hydroxyalkyl groups, and which may be further fused to a carbocyclic ring, or R13 and R15 are each hydrogen and R17, together with R18, forms a fused fully or partially saturated non-aromatic cyclohexyl ring as defined above, or R13, together with R15, forms a fused fully or partially saturated non-aromatic cyclohexyl ring as defined above and R17 and R18 are hydrogen; provided that (a.) when G is always xe2x80x94COORxe2x80x3 and (i.) Rxe2x80x2, Rxe2x80x3, R14 and R16 through R20 are all hydrogen, then R13 and R15 are other than hydrogen; (ii.) Rxe2x80x3 and R13 through R20 are all hydrogen, and m, n, o, p and q are each 1, then (CRxe2x80x2Rxe2x80x2) is other than (CH2) and (CHCH3); (iii.) Rxe2x80x2, Rxe2x80x3, R13, R14, R17 and R20 are all hydrogen, then at least two of R15, R16, R18 and R19 are other than methyl; and (iv.) Rxe2x80x3, R16, R19 and R20 are all hydrogen, and each (CRxe2x80x2Rxe2x80x2) is independently (CHRxe2x80x2) or (CH2CHRxe2x80x2), then R13 and R15, and R17 and R18, are other than a fused ring; and (b.) when G is always xe2x80x94P(O)(ORxe2x80x3)2, xe2x80x94P(O)(ORxe2x80x3)(Rxe2x80x3) or xe2x80x94C(O)N(Rxe2x80x3)2, then at least one Rxe2x80x2 or R13 through R20 is other than hydrogen;
or a salt or multimeric form thereof.
Listed below are definitions of various terms used in the description of this invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.
The expression xe2x80x9crelaxivityxe2x80x9d refers to the effectiveness of a metal chelate to reduce the relaxation time of bulk water in contact with the metal chelate.
The expression xe2x80x9cimmobilized relaxivityxe2x80x9d refers to the relaxivity measured when a chelate moiety can undergo only slow molecular reorientation because of rigid attachment to a large moiety or a physiological surface, or because it is dissolved in a medium of high viscosity.
The expression xe2x80x9cwater relaxivityxe2x80x9d refers to relaxivity in water where a chelate moiety possesses a relaxivity dominated by overall molecular reorientation.
The expression xe2x80x9cenhanced relaxivityxe2x80x9d refers to relaxivity values made greater than those of well characterized prior art molecules by 1.) altering the electronic relaxation rate, xcfx84s, through modifications of the metal-donor atom bond vibration frequencies and/or amplitudes, (this being accomplished, for example, by increasing the steric bulk and/or orientation of organic elements bonded to the donor atoms and/or the macrocyclic carbon atoms), 2.) in a multimer by decreasing the internal molecular motion of one monomer unit relative to another (this being accomplished, for example, by increasing the steric bulk of the organic groups linking the monomer units) or 3.) by decreasing the molecular reorientation of a monomer or a multimer attached to a large moiety or a physiological surface.
The term xe2x80x9cstabilityxe2x80x9d refers to the equilibrium formation constant (K) of the reaction M+Lxe2x86x92M(L) where K=[M(L)]/[M][L], M is a metal ion, L is a chelating ligand and M(L) is a chelate complex of a metal and a ligand.
The term xe2x80x9calkylxe2x80x9d refers to both straight and branched, unsubstituted chains of carbon atoms. Those chains having 1 to 5 carbon atoms are preferred. Methyl is the most preferred alkyl group.
The term xe2x80x9ccycloalkylxe2x80x9d refers to cyclic hydrocarbon groups of 3 to 8 carbon atoms. The groups may be unsubstituted or substituted by, for example, alkyl, halogen, hydroxy, hydroxyalkyl, alkoxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, alkanoylamino, thiol, alkylthiol, nitro, cyano, carboxy, carbamoyl, alkoxycarbonyl, alkylsulfonyl, sulfonamido and the like.
The term xe2x80x9calkoxyxe2x80x9d refers to -alkyl(O). Methoxy is the most preferred alkoxy group.
The term xe2x80x9carylxe2x80x9d refers to phenyl, pyridyl, furanyl, thiophenyl, pyrrolyl, imidazolyl and the like, all of which may be substituted. Preferred substituted aryl groups are those substituted with 1, 2 or 3 halogen, nitroamino, maleimido, isothiocyanato, hydroxy, hydroxyalkyl, alkyl, alkoxy, carbamoyl, carboxamide, acylamino or carboxy moieties.
xe2x80x9cHydroxyalkylxe2x80x9d refers to straight and branched alkyl groups including one or more hydroxy radicals such as xe2x80x94CH2CH2OH, xe2x80x94CH2CH2OHCH2OH, xe2x80x94CH(CH2OH)2 and the like. (See, for example, Sovak, M., Editor, Radiocontrast Agents, Springer-Verlag, 1984, pp. 1-125).
The term xe2x80x9caralkylxe2x80x9d refers to an aryl group bonded through an alkyl group.
The term xe2x80x9ccarbocyclic ringxe2x80x9d refers to a ring system in which all the ring atoms are carbon, e.g., phenyl or cyclohexyl. The ring may be unsubstituted or substituted by, for example, alkyl, halogen, hydroxy, hydroxyalkyl, alkoxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, alkanoylamino, thiol, alkylthiol, nitro, cyano, carboxy, carbamoyl, alkoxycarbonyl, alkylsulfonyl, sulfonamido and the like.
The term xe2x80x9chalogenxe2x80x9d refers to bromo, chloro, fluoro or iodo.
The term xe2x80x9calkanoylxe2x80x9d refers to the group alkyl-C(O)xe2x80x94.
The term xe2x80x9calkanoyloxyxe2x80x9d refers to the group alkyl-C(O)xe2x80x94Oxe2x80x94.
The term xe2x80x9caminoxe2x80x9d refers to the group xe2x80x94NH2.
The term xe2x80x9calkylaminoxe2x80x9d refers to the group xe2x80x94NHR where R is alkyl.
The term xe2x80x9cdialkylaminoxe2x80x9d refers to the group xe2x80x94NRRxe2x80x2 where R and Rxe2x80x2 are each, independently, alkyl.
The term xe2x80x9calkanoylaminoxe2x80x9d refers to the group alkyl-C(O)xe2x80x94NHxe2x80x94.
The term xe2x80x9cthiolxe2x80x9d refers to the group xe2x80x94SH.
The term xe2x80x9calkylthiolxe2x80x9d refers to the group xe2x80x94SR where R is alkyl.
The term xe2x80x9cnitroxe2x80x9d refers to the group xe2x80x94NO2.
The term xe2x80x9ccyanoxe2x80x9d refers to the group xe2x80x94CN.
The term xe2x80x9ccarboxyxe2x80x9d refers to the group xe2x80x94C(O)OH or the group xe2x80x94C(O)OR where R is alkyl.
The term xe2x80x9calkoxycarbonylxe2x80x9d refers to the group alkoxy-Cxe2x80x94(O)xe2x80x94.
The term xe2x80x9calkylsulfonylxe2x80x9d refers to the group alkyl-SO2xe2x80x94.
The term xe2x80x9csulfonamidoxe2x80x9d refers to the group xe2x80x94SO2NH2, the group xe2x80x94SO2NHR or the group xe2x80x94SO2NRRxe2x80x2 where R and Rxe2x80x2 are each, independently, alkyl, cycloalkyl or aryl.
The term xe2x80x9ccarbamoylxe2x80x9d refers to the group xe2x80x94C(O)NH2, the group xe2x80x94C(O)NHR or the group xe2x80x94C(O)NRRxe2x80x2 where R and Rxe2x80x2 are each, independently, alkyl, alkoxy or hydroxyalkyl.
The term xe2x80x9ccarboxamidexe2x80x9d refers to the group xe2x80x94C(O)NH2, the group xe2x80x94C(O)NHR or the group xe2x80x94C(O)NRRxe2x80x2 where R and Rxe2x80x2 are each, independently, alkyl.
The term xe2x80x9cacylaminoxe2x80x9d refers to the group xe2x80x94NHxe2x80x94C(O)xe2x80x94R where R is alkyl.
The expressions xe2x80x9cbioactive groupxe2x80x9d and xe2x80x9cbioactive moietyxe2x80x9d denote a group which is capable of functioning as a metabolic substrate, catalyst or inhibitor, or is capable of being preferentially taken up at a selected site of a subject, such as by possessing an affinity for a cellular recognition site.
When compounds of the formula I are in the multimeric form, each monomer is preferably linked by a cyclic bridging group represented by the general formula II: 
wherein
Q is a 4- to an 8-membered carbocyclic ring which may be fully or partially saturated;
t is an integer from 2 to 16;
each R group is independently hydrogen, xe2x80x94OH, xe2x80x94CH2xe2x80x94A, xe2x80x94OCH2CH(OH)CH2xe2x80x94A or a functional group capable of forming a conjugate with a biomolecule, provided that at least two of the R groups are selected from xe2x80x94CH2xe2x80x94A or xe2x80x94OCH2CH(OH)CH2xe2x80x94A; and
A is the monomer of formula I.
Of course, the cyclic bridging group may be used to link known moieties as well. Where it is a known moiety which is being linked by the cyclic bridging group of formula II, A is any moiety capable of chelating a metal atom.
Preferred bridging groups of the formula II are those wherein 
is a compound of the following formula III: 
wherein
each R1 through R12 group is independently hydrogen, xe2x80x94OH, xe2x80x94CH2xe2x80x94A, xe2x80x94OCH2CH(OH)CH2xe2x80x94A or a functional group capable of forming a conjugate with a biomolecule;
at least two of R1 through R12 are selected from xe2x80x94CH2xe2x80x94A or xe2x80x94OCH2CH(OH)CH2xe2x80x94A;
R8 and R9 taken together may additionally form the group xe2x80x94Oxe2x80x94[C(RR)]xe2x80x94Oxe2x80x94 where each R is independently hydrogen or alkyl, or R8 and R9 taken together may form 
xe2x80x83and
A is a moiety described above.
When A is a known monomer, for example, A is preferably 
wherein
each Rxe2x80x2 is independently hydrogen, alkyl, alkoxy, hydroxyalkyl, aryl, aralkyl or arylalkoxy;
each Rxe2x80x3 is hydrogen; and
each n is 1 or 2;
or a salt thereof.
Contrast agents with significantly enhanced relaxivities are of great interest, not only because they offer improved efficacy at reduced doses for current clinical applications, but also because they may provide the sensitivities needed for imaging various biochemical processes.
Certain preferred compounds having enhanced relaxivities are (i.) chelates possessing a stability greater than or equal to 1015 Mxe2x88x921 and capable of exhibiting an immobilized relaxivity between about 60 and 200 mMxe2x88x921sxe2x88x921/metal atom, for example between about 70 and 150 mMxe2x88x921sxe2x88x921/metal atom, or between about 80 and 100 mMxe2x88x921sxe2x88x921/metal atom; and (ii.) multimeric chelates possessing monomer units with a stability greater than or equal to 1015 Mxe2x88x921, and having relaxivity values (not immobilized) greater than 5 mMxe2x88x921sxe2x88x921/metal atom.
In designing new chelates with these elevated relaxivities, the inventors have noted that immobilized relaxivity can depend strongly, for example, on the structure of the chelate. Without being bound by any particular theory, apparently a mechanism that involves rigidifying the chelate structures in solution with, for example, alkyl substitutions on the tetraaza ring of chelates, especially when the substitution is introduced into the carboxylate arms of the chelates, affects immobilized relaxivity. In the Solomon-Bloembergen-Morgan (SBM) model, the electronic relaxation of a paramagnetic metal complex is viewed as occuring through a dynamic modulation process about the transient zero-field splitting (ZFS) of the metal""s electronic spin levels. This transient ZFS is induced by the structural distortion of the metal complex from its ideal symmetry in solution which is thought to be caused by its collision with solvent molecules. Alkyl substitution on either the tetraaza ring or the carboxylate arms of a chelate is thought to reduce the flexibility of the chelate for structural distortion in solution. This in turn reduces the magnitude of ZFS, giving an increased immobilized relaxivity value.
Water relaxivity is generally determined in aqueous Bis Tris buffer (pH 7) solutions by the standard inversion-recovery method (known to those in the art) at 20 MHz and 40xc2x0 C. (See, e.g., X. Xhang, Inorganic Chemistry, 31, 1992, 5597, the entire contents of which are hereby incorporated by reference.) While a mathematical description of the relaxation mechanism at the presence of a paramagnetic species is provided by the classical SBM equations, it is experimentally difficult to explore the dependence of relaxivity on structure because the relaxivity values of most low-molecular weight chelates are often controlled by their rapid tumbling motions in regular aqueous solutions.
To eliminate the overriding effect of molecular tumbling in regular aqueous solutions, the relaxivity of a chelate can be determined in aqueous sucrose solutions or other media that result in a reduction of molecular reorientation of any solute. In these solutions, the relaxivity values approximate, under optimal conditions, the relaxivities of chelates in biologically immobilized systems such as those covalently attached to cell surfaces. Hence, these values in sucrose solutions are defined as immobilized relaxivity. Immobilized relaxivity is generally determined under the same conditions as water relaxivity (see supra) except that the viscosity of the solutions is increased to 80 cp by the addition of solid sucrose to the aqueous solution of chelate, and the temperature is set at 20xc2x0 C. In the calculation of relaxivity, one uses the concentration of the solute in the aqueous solution (before sucrose is added).
When used for relative comparison, this method serves as a simple screening technique for differentiating chelates designed for efficient biological targeting.
Thus, the compounds of the invention, including compounds of the formula I and formula II, and salts thereof, may be complexed with a paramagnetic metal atom and used as relaxation enhancement agents for magnetic resonance imaging. These agents, when administered to a mammalian host (e.g., a human) distribute in various concentrations to different tissues, and catalyze relaxation of protons (in the tissues) that have been excited by the absorption of radiofrequency energy from a magnetic resonance imager. This acceleration of the rate of relaxation of the excited protons provides for an image of different contrast when the host is scanned with a magnetic resonance imager. The magnetic resonance imager is used to record images at various times, generally either before and after administration of the agents, or after administration only, and the differences in the images created by the agents, presence in tissues are used in diagnosis. In proton magnetic resonance imaging, paramagnetic metal atoms such as gadolinium(III), and manganese(II), chromium(III) and iron(III) (all are paramagnetic metal atoms with favorable electronic properties) are preferred as metals complexed by the ligands of the invention, including the ligands of formula I and formula II. Gadolinium(III) is the most preferred complexed metal due to the fact that it has the highest paramagnetism, it has low toxicity when complexed to a suitable ligand, and it has high lability of coordinated water. When the distance between the monomeric gadolinium chelate units in a complex is at least about 6 angstroms, the complexes tend to be sufficiently stable. Those compounds of formula II, when complexed with gadolinium(III) ions, are particularly useful. The distance between the monomeric gadolinium chelate units in these complexes is generally greater than 6 angstroms (although in certain circumstances a distance of 4.5 angstroms is sufficient), and the rigid bridges of these complexes assist in reducing independent motion of the gadolinium ions.
The metal-chelating ligands of the present invention can also be complexed with a lanthanide (atomic number 58 to 71) and used as chemical shift or magnetic susceptibility agents in magnetic resonance imaging or in magnetic resonance in vivo spectroscopy.
While the above-described uses for the metal-chelating ligands of the present invention are preferred, those working in the diagnostic arts will appreciate that the ligands can also be complexed with the appropriate metals and used as contrast agents in other imaging techniques such as x-ray imaging, radionuclide imaging and ultrasound imaging, and in radiotherapy.
To use the ligands of the present invention for imaging, they are first complexed with an appropriate metal. This may be accomplished by methodology known in the art. For example, the metal can be added to water in the form of an oxide or in the form of a halide or acetate and treated with an equimolar amount of a ligand of the present invention. The ligand can be added as an aqueous solution or suspension. Dilute acid or base can be added (where appropriate) to maintain a suitable pH. Heating at temperatures as high as 100xc2x0 C. for periods of up to 24 hours or more may sometimes be employed to facilitate complexation, depending on the metal and the chelator, and their concentrations.
Pharmaceutically acceptable salts of the metal complexes of the ligands of this invention are also useful as imaging agents. They can be prepared by using a base (e.g., an alkali metal hydroxide, meglumine, arginine or lysine) to neutralize the above-prepared metal complexes while they are still in solution. Some of the metal complexes are formally uncharged and do not need cations as counterions. Such neutral complexes may be preferred in some situations as intravenously administered x-ray and NMR imaging agents over charged complexes because they may provide solutions of greater physiologic tolerance due to their lower osmolality.
The present invention also provides pharmaceutical compositions comprising a compound of the invention, including a compound of the formula I or II, or a salt of one of these compounds, optionally complexed with a metal, and a pharmaceutically acceptable vehicle or diluent. The present invention further provides a method for diagnostic imaging comprising the steps of administering to a host a compound of the invention, or a salt thereof, which is complexed with a metal, and obtaining a diagnostic image, preferably a magnetic resonance image, of said host.
Sterile aqueous solutions of the chelate complexes of the present invention are preferably administered to mammals (e.g., humans) orally, intrathecally and, especially, intravenously in concentrations of about 0.003 to 1.0 molar. The metal complexes of the present invention may be employed for visualization of various sites. For example, for the visualization of brain lesions using magnetic resonance imaging, a gadolinium complex of a ligand of the invention, including a ligand of the formula I or formula II, may be administered intravenously at a dose of 0.001 to 0.5 millimoles of the complex per kilogram of body weight, preferably at a dose of 0.001 to 0.3 millimoles/kilogram. For visualization of the kidneys, the dose is preferably 0.05 to 0.20 millimoles/kilogram. For visualization of the heart, the dose is preferably 0.001 to 0.3 millimoles/kilogram. For visualization of the liver, the dose is preferably 0.001 to 0.3 millimole/kilogram.
The pH of the formulation of the present metal complexes is preferably between about 6.0 and 8.0, most preferably between about 6.5 and 7.5. Physiologically acceptable buffers (e.g., tris(hydroxymethyl)-aminomethane) and other physiologically acceptable additives (e.g., stabilizers such as parabens) may also be present.
It is also advantageous to employ dual scavenging excipients such as those described in copending application U.S. Ser. No. 032,763, filed Mar. 15, 1993, entitled xe2x80x9cDUAL FUNCTIONING EXCIPIENT FOR METAL CHELATE CONTRAST AGENTSxe2x80x9d, incorporated herein by reference. Those excipients have a general formula corresponding to:
Ds[Dxe2x80x2(Lxe2x80x2)]t
wherein D and Dxe2x80x2 are independently Ca or Zn, Lxe2x80x2 is an organic ligand which may be different from, or the same as, the ligand employed to complex the metal, and s and t are independently 1, 2 or 3.
As already noted, the present invention further includes multimeric forms of the compounds of the invention, including those of formula I and formula II, such as dimers, trimers, tetramers, etc. Known functional groups and technology are readily useable to provide such multimers.
Compounds of the present invention may include those containing functional group(s) capable of forming a conjugate with a biomolecule. These compounds are preferably chelates, including a functional group, capable of exhibiting an immobilized relaxivity between about 60 and 200 mMxe2x88x921sxe2x88x921/metal atom, for example between about 70 and 150 mMxe2x88x921sxe2x88x921/metal atom, or between about 80 and 100 mMxe2x88x921sxe2x88x921/metal atom. Similarly, the chelates, once conjugated to a biomolecule of size greater than or equal to about 40,000 daltons, are also preferably capable of exhibiting a relaxivity between about 60 and 200 mMxe2x88x921sxe2x88x921/metal atom, for example between about 70 and 150 mMxe2x88x921sxe2x88x921/metal atom, or between about 80 and 100 mMxe2x88x921sxe2x88x921/metal atom. Preferred biomolecules are peptides, polypeptides and oligosaccharides or fragments thereof, although other biomolecules such as proteins, particularly monoclonal antibodies, lipids, sugars, alcohols, bile acids, fatty acids, receptor-binding ligands, amino acids and RNA, DNA or modified fragments of these may be conjugated to the compounds of the present invention. For smaller biomolecules, the enhanced relaxivity afforded by the chelates of this invention may be more fully realized when the chelate-biomolecule conjugate becomes immobilized in vivo, such as by binding to a receptor on a cell surface or by binding to another biomolecule.
Conjugates where a compound of the invention, or salt and/or multimer thereof, is linked to a biomolecule such as a protein, provided by the present invention, are novel, as are metal complexes and pharmaceutical compositions containing, and methods of using (e.g., for imaging), the aforementioned conjugates. Conjugation may be achieved in vitro, such as by use of known conjugation methodologies, or in situ in a living subject by administration of a compound containing one or more of the aforementioned functional groups.
For linking the compounds of the present invention to a protein, the R groups may be reacted with a protein to produce a protein conjugate. Preferred proteins are those in serum, wherein the compound of the invention is directly injected and the conjugate is formed in situ. It is understood that other functional groups, as described above, may be used to link the bifunctional metal-chelating ligands of this invention to proteins such as monoclonal antibodies or fragments thereof.
Compounds of the formula I can generally be prepared as follows:
Ligands in which the aza macrocyclic ring carbon atoms are modified are built de novo from suitable aziridine precursors by cyclotetramerization.
Cyclotetramerization of N-benzylaziridine has been reported in the literature. (See, for example, T. E. Burg and G. R. Hansen, J. Heterocyclic Chemistry, (1968), 5, 305.) The synthetic approach to the preparation of S,S,S,S-tetramethyltetraazacyclododecane (5) is given in scheme 1: 
[S]-N-Benzoylalanine(1) is reduced with diborane to yield [S]-N-benzylalaninol(2). Under Mitsunobu conditions, compound (2) affords [S]-N-benzyl-2-methyl aziridine(3). Cyclotetramerization under p-toluene sulfonic acid catalysis in ethanol, followed by treatment with ammonium hydroxide (NH4OH), furnishes S,S,S,S-tetra-N-benzyltetramethyltetraazacyclododecane(4), which is debenzylated under transfer hydrogenolytic conditions to obtain S,S,S,S-tetramethyltetraazacyclododecane(5).
Tetraalkylation of compound (5) with t-butyl bromoacetate in the presence of sodium carbonate, followed by deprotection with trifluoroacetic acid and anisole, affords the ligand (6).
Tetraalkylation of compound (5) with benzyl 2-triflyloxylactate (prepared as described by S. I. Kang et al., Inorg. Chem., (1993), 32, 2912-2918) in the presence of sodium carbonate, followed by catalytic hydrogenolytic debenzylation furnishes the ligand (7).
Tris-alkylation of compound (5) with t-butyl bromoacetate in the presence of sodium bicarbonate, followed by deprotection by treatment with trifluoroacetic acid and anisole, provides the ligand (8).
For preparing multimeric ligands, 1,4,7-tris-carboxymethyl-1,4,7,10-tetraazacyclododecane-10-yl (DO3A) units, for example, are attached to a bridging unit by two different methods. In the first method, a spiro epoxide on the bridging unit is used to alkylate DO3A. In the second method, a glycidyloxy moiety is attached to the bridging unit which then is used to alkylate DO3A.
By way of example, N-alkylation of DO3A with suitable epoxides of formula (9) below, wherein X is a carbocyclic, fused heterocyclic or spiro-heterocyclic unit, and n is 2, 4 or 8, affords multimeric ligands of the formula (10): 
Epoxides of formula (9) are generated by the Prilezhaev Reaction in which olefins of formula (11) are treated with peracids such as m-chloroperbenzoic acid: 
Olefins of formula (11) are generated by the alkylation of alcohols of formula (12): 
Another mode of attachment of DO3A would be to epoxidize olefins of formula (13) to obtain epoxides of formula (14), and then alkylating DO3A with the epoxides to form ligands of formula (15): 
These two method of attachment of DO3A could also be present in the same bridging unit X leading to structures as in formula (16): 
Representative bridging units are exemplified in formulas (17), (18), (19), (20), (21) and (22): 
The structures shown in formulas (17) to (22) are only by way of representative examples. They will, in actuality, be a mixture of all the possible diastereomers, if the attachment is through a 2-hydroxypropyloxy link. In the case of formula 22, for example, in addition to the presence of diastereomers, the product would also consist of the various geometric isomers that could result when the two myo-inositol molecules are coupled to each other. The coupling of the two myo-inositol units is achieved by reacting the compound of formula (23) with cyclohexane-1,4-dione (24): 
The bridging unit for formula 17 is generated from commercially available diethyl cyclohexane-1,4-dione-2,5-dicarboxylate by lithium aluminum hydride reduction as described in J. G. Murphy, J. Med. Chem., (1966), 9, 157.
The bridging unit for formula (18) is generated by treating myo-inositol (25) with 2,2-dimethoxypropane in the presence of p-toluene sulfonic acid as descirbed by Giggs et al., Carbohydrate Res., 1985, 1, 132: 
The bridging unit (19) is made from (18) by acidic hydrolysis. The unit (20) is prepared from myo-inositol (25) by reaction with excess 2,2-dimethoxypropane in the presence of p-toluenesulfonic acid. The unit (21) is made from the bis-epoxide precursor to the unit (17) by methods desribed above for functionalizing hydroxy groups.
The ligand of Example (10), below, bearing a phosphonomethyl arm, is made by treating DO3A with phosphorus acid and formaldehyde as described by M. Tazaki et al., Chem. Lett., 1982, 571.
All stereoisomers of the compounds and complexes of the present invention are contemplated herein, whether alone (that is substantially free of other isomers), in a mixture of certain stereoisomers (for example, as a racemate) or in any other mixture thereof.